Advancements in precise genetic manipulation have helped biologists to identify new drug-targets and in vivo disease mechanisms using small animal models, such as C. elegans. Human diseases pathophysiologies are reproduced in C. elegans expressing human disease genes inside the animal. Great opportunities are provided by the recent surge is genetic tools for animal models, but the lack of high-content screening (HCS) technologies precluded these models from screening for subtle phenotypes that better recapitulate the human disease situations. Development of novel technologies will enable the use of such models, at the same cost and speed of in vitro assays, to discover new drug targets and understand mode-of-action for new compounds in vivo. Dr. Ben-Yakar Lab at The University of Texas at Austin has developed a novel large-scale microfluidic chip that can image ~4,000 animals from 96 populations using a proprietary channel design. An efficient image acquisition and analysis algorithms can screen a whole chip within 16 min, a record speed that is 10,000 faster than manual imaging. To translate this lab prototype into marketplace, this SBIR Phase I application proposes to develop a beta model vivoChip-96x that will be with SBS format, compatible with automation, lighter weight, less expensive, and user-friendly to operate with the new top-gasket design. The new chip design will be bonded to a thin glass substrate at the bottom to enable improved imaging using high-resolution objectives. The proposed microfluidic chip will incorporate a machined top-plastic with custom-designed wells in a micro-titer format for easy integration to liquid handling systems for the high-throughput screen (HTS), and avoid substrate bending, and chip-handling errors. In Aim 1, we plan to develop a beta design of the vivoChip-96x device with the top- plastic piece, fabricate a thin PDMS layer using soft-lithography, and bond the interfaces using appropriate UV- cured glue and plasma treatment. The beta model will be operated with a new top-gasket with an improved sealing mechanism. C. elegans populations will be trapped inside the channels to characterize the variability in the trapping efficiency using four chips and following the standard operational procedures (SOPs). In Aim 2, we will develop an automated acquisition algorithm with BioTek to image C. elegans with high reslution objectives having a sub-cellular expression of fluorescent proteins and achieve an assay quality Z?~0.8. Achieving these milestones in Phase I, we will be able to reduce the current cost of the chip by 3 folds and standardize the vivoChip-96x for all commercially available HCS instruments. In Phase II, we will develop a fully automated vivoLoader to replace our current semi-automated worm handling procedures of liquid handling and an automated image-analysis platform (vivoAnalyzer) that will identify subtle fluorescent phenotype in low expressing C. elegans. Using our ongoing research collaboration, we plan to apply our screening technology to develop neurotoxicity and neurodegeneration assays to be able to screen novel compounds from large pharmaceutical companies. Support from industry partners will help us to translate the prototype into a product.